SUMMARY: Acquired epilepsy is a chronic condition that requires life-long medication and is often drug- resistant. The development of more efficient drugs to prevent or reduce epilepsy is currently hindered by a gap in knowledge of how an epilepsy-precipitating event turns a healthy brain into a brain that produces spontaneous recurrent seizures. This process, known as epileptogenesis, is thought to be highly complex. Halting or preventing epileptogenesis thus most likely requires the concerted manipulation of many different molecular networks and pathways. It is therefore plausible that treatment strategies targeting regulatory mechanisms that control multiple of these cellular pathways at once will be most successful. This research will address this challenge by analyzing how a potent regulator of the expression of hundreds of genes, the microRNA-induced silencing complex (RISC), contributes to epileptogenic processes after status epilepticus (SE). Previous work supports a role of microRNAs and the RISC in epilepsy development by showing that select microRNAs and mRNAs are recruited to the RISC after seizure, and that inhibition of single microRNAs reduces seizure susceptibility and epileptogenesis in rodent epilepsy models. Yet, the molecular mechanisms and pathways underlying the seizure-mitigating effects of microRNA manipulation are largely unknown. The overall hypothesis of this research is that SE induces changes in RISC and microRNA function that enhance epileptogenic and decrease neuroprotective pathways. Inhibiting these changes may prevent or impair the development of epilepsy. This hypothesis will be tested with two aims. Aim 1 will follow an unbiased approach using cell type- specific immunoprecipitation of the RISC, RNA sequencing and genetic knockdown strategies to reveal the nature and functional relevance of molecular pathways that are differentially regulated by the RISC after SE in mice. Aim 2 will follow a candidate-based approach using ribosomal tagging and functional assays together with antisense-mediated microRNA inhibition to reveal the cell-specific translatome of a pro-convulsive microRNA and how it contributes to epileptogenesis after SE. Based on complementing expertise of neuroscientists and computational biologists, the approach to perform screens of RISC and microRNA target regulation after SE paired with pathway and functional analyses is expected to generate unique information about the complex processes regulating epileptogenesis. The innovative strategy using RISC association as a surrogate for microRNA function, and association of mRNAs with the RISC or actively translating ribosomes as a surrogate for their silencing or translation, respectively, is expected to provide an improved functional assessment of the silencing activity of microRNAs and the effect on target mRNAs compared to previous expression analyses. This study will fill a crucial gap in the understanding of RISC function, protein expression and pathway dysregulation in epileptogenesis, which will be vital to advance microRNA-induced silencing as therapeutic target. Seen from a broader perspective, this strategy could serve as a blueprint for RISC analysis in other diseases.